Evaluating Small Molecule Cell Cycle and Chromatin Condensation Modulators Using a Novel Green
Fluorescent DNA-Binding Probe: Potential Application to Compound Screening
ID 499; P47
Nyaya Kelkar, Zaiguo Li, Irina Lebedeva, Dee Shen, Praveen Pande and Wayne F. Patton; Enzo Life Sciences, Farmingdale, NY 11735
ABSTRACT
When eukaryotic cells undergo replication, they pass through a tightly regulated series of events known as the cell
cycle, marked by distinctive characteristics as the DNA is replicated. Cell cycle checkpoints at specific points in the
process prevent cells from progressing to the subsequent phase of the cell cycle in the event of DNA damage or other
adverse conditions that might impact overall survival. Considerable progress has been made in the analysis of the
stages of cell cycle progression, with a number of cell permeable small molecules identified to modulate the process.
Flow cytometry is a commonly implemented platform for cell cycle analysis, but the most widely employed dye for the
instrument, propidium iodide, requires permeabilization or fixation of cells. A number of newer cell-permeable dyes
have subsequently been introduced but most require very restrictive staining conditions and laborious standardization
of techniques. We have introduced a novel fluorescent probe that facilitates cell cycle analysis in live cells. The green
fluorescent probe can be used in a mix and read format over a wide concentration range (5-20 µM) employing a wide
range of cell densities (1x 105-1x106 cells/ml). Moreover the dye provides substantial flexibility with respect to the
incubation medium, time and temperature used in the analysis. Live cell cycle analysis was benchmarked using a panel
of 12 small molecule cell cycle modulators known to perturb cells at the G0/G1, S or G2/M phases in a concentrationdependent manner. Apoptotic cell death was monitored as well through protocol modifications allowing sub G0 analysis
and chromatin condensation determination. The described fluorescent probe should be applicable to the analysis of
the phases of the cell cycle, especially as applied to the identification of small molecule modulators for use in treatment
strategies targeting cell cycle checkpoints.
105 cells/ml
5 x 105 cells/ml
%G1 = 28.5
%S = 26.5
%G2 = 44.9
%G1 = 43.2
%S = 32.7
%G2 = 22.4
Number of
cells
%G1 = 47.7
%S = 7.7
%G2 = 45.8
106 cells/ml
Control
Incubation
time
15 min
%G1 = 27
%S = 55
%G2 = 29
30 min
Aloisine 20 µM
%G1 = 13.3
%S = 10.2
%G2 = 76.9
Trichostatin A 250 nM
%G1 = 10
%S = 10
%G2 = 80
Neoxaline 70 uM
%G1 = 9
%S = 7
%G2 = 85
Control
1.25 µM
2.5 µM
5 µM
7.5 µM
10 µM
Control
1.25 µM
2.5 µM
3.75 µM
5 µM
10 µM
%G1 = 15.24
%S = 14.6
%G2 = 70.7
60 min
Monastrol 25 µM
FIGURE 2: Cell cycle results independent of cell concentration and incubation time.
Vinblastine 10 µM
Nocodazole 0.1 ng/ml
TN16 100 ng/ml
FIGURE 8: Alosine and chromatin condensation.
FIGURE 5: Molecules affecting G1/G2 Phases.
FIGURE 11: Vinblastine and chromatin condensation.
INTRODUCTION
Monitoring Chromatin Condensation in a 96-well Microplate
3000
Alosine
RT
37° C
Thus the Nuclear-ID™ Green probe is suitable for (1) Analysis of cell cycle modulation by various drugs and (2)
differentiating between healthy and apoptotic cells with condensed nuclei on various instrument platforms.
70
60
G1
S
G2
20 µM
G1
30
1500
S
20
G2
1000
0
Control
2.5 uM
5 uM
10 uM
20 uM
0.62
ug/ml
40 uM
1.25
ug/ml
2.5 ug/ml
3.75
ug/ml
5 ug/ml
Control
0.62 µM
1.25 µM
500
Vinblastine
80
70
70
60
0
Control
% Cell Count
50
G1
40
S
30
G2
40
G1
30
S
10
0
0
Control
1.25 uM
2.5 uM
5 uM
A2
A3
B1
B2
B3
C1
C2
C3
G2
20
10
A1
FIGURE 12: Chromatin Condensation Micro-Plate Assay. There is increase in the relative fluorescence values upon treatment with
various drugs for 4 hours; Compound A, Camptothecin (1,2 and 5 µM) does not show condensation in given conditions; Compounds B,
BML-258 (10,20 and 50 µM) and C, Staurosporine, (0.5, 1 and 2 µM) show concentration-dependent dose-response profiles..
50
20
10 µM
40
10
60
5 µM
2000
50
Etoposide
Dye
concentration
% Cell Count
Several small molecules permeable to cells are known that affect the progression of cell cycle in a specific manner. We
have examined the effect of a range of such molecules, available from Enzo Life Sciences, on the phases of cell cycle
as well as cellular health.
50
45
40
35
30
25
20
15
10
5
0
Control
Additionally, Nuclear-ID™ Green probe provides a convenient approach for analysis of late stage apoptosis by flow
cytometry and fluorescence microscopy. When incubated with Nuclear-ID™ Green probe, the compacted chromatin of
apoptotic cells take up increased amounts of stain compared to the healthy cells. The Nuclear-ID™ Green probe can be
used in microplate assays in HTS format as well.
2500
Aphidicolin
% Cell Count
Incubation
temperature
% Cell Count
The progression of the cell cycle is controlled by a complex interplay among various cell cycle regulators that either
stimulate or inhibit the cell from entering each stage of the cell cycle. Dysfunction of any step in this regulatory cascade
causes abnormal cell proliferation which underlies many human pathological conditions, such as cancer. A crucial
step to understanding these conditions is the ability to understand the mechanisms underlying alterations in cell cycle
progression. Enzo Life Sciences’ Nuclear-ID™ Green probe provides a convenient approach for studying the induction
and inhibition of cell cycle progression by flow cytometry. It is suitable for (1) determining the percentage of cells in a
given sample that are in G0/G1, S and G2/M phases, as well as to quantify cells in the sub-G1 phase, and (2) DNA studies
in live, permeabilized and fixed cells for normal cell lines and cell lines exhibiting multiple ploidy levels. The green probe
also provides flexibility with respect to (1) sample size, (2) medium for staining, (3) concentration of probe for staining
and (4) incubation temperature.
10 uM
Control
1.25 uM
2.5 uM
3.75 uM
5 uM
10 uM
2.5 µM
3.75 µM
5 µM
CONCLUSIONS
FIGURE 3: Cell cycle results independent of incubation temperature and dye concentration.
FIGURE 9: Aphidicolin and chromatin condensation.
FIGURE 6: Cellular dose response to selected compounds.
• Nuclear-ID™ Green dye has an absorption maximum of 504 nm and emission maximum of 531 nm, making
it compatible with any instrument that can detect FITC.
• The dye readily stains live, permeabilized or fixed cells.
• Nuclear-ID™ Green dye can efficiently be used for cell cycle analysis.
%G1 = 40.8
%S = 31.1
%G2 = 24.6
Fluorescence Properties
%G1 = 39.5
%S = 46.9
%G2 = 12.7
• Nuclear-ID™ Green dye can be used to study molecules affecting cell cycle progression
Chromatin Condensation as Detected by Fluorescence Microscopy
• Nuclear-ID™ Green dye detects changes in chromatin structure arising from apoptosis as a ~50-fold increased
fluorescence in the apoptotic nuclei.
2500
Z' >0.75
2000
1500
Control
1.25 µM
%G1 = 6
%S = 63
%G2 = 30
%G1 = 43.4
%S = 48.6
%G2 = 11.1
Product
500
Staurosporine
0
0.0
400
450
500
Wavelength (nm)
FIGURE 1: Absorption-Emission Spectra of Nuclear-ID™ Green Dye
550
Reagents and Kits used in This Study
2.5 µM
1000
0.5
1.0
1.5
2.0
2.5
3.0
Valinomycinn Con (mM)
Blank Filter
600
Etoposide
FIGURE 4: Molecules affecting S phase.
3.5
4.0
4.5
FITC Filter
Quercetin 25 µM
3.75 µM
FIGURE 7: Chromatin condensation as observed by fluorescence microscopy. HeLa cells were treated for 4 hours with DMSO (Control)
or 2 µM Staurosporine on a slide and stained with 5 µM Nuclear-ID™ Green dye.
FIGURE 10: Etoposide and chromatin condensation.
5 µM
10 µM
Prod. No.
Nuclear-ID™ Green Cell Cycle Kit for flow cytometry
ENZ-51014-100
Nuclear-ID™ Green Chromatin Condensation Detection Kit for fluorescence microscopy and flow cytometry
ENZ-51021-K200
Alosine A
ALX-270-385
Aphidicolin
BML-CC101
Hesperetin
ALX-385-011
Monastrol
GR-322
Neoxaline
ALX-350-409
Quercetin dihydrate
ALX-385-001
Vinblastine
ALX-350-257
TN-16
T-120
Trichostatin A
GR-309
Nocodazole
BML-T101
Etoposide
BML-GR307
www.enzolifesciences.com
ZZ-Sc0510-1009
Hesperetin 60 µM
RFU
Absorbance
Fluorescence emission
Control
Control